Antisense compounds contain oligonucleotides that bind to or hybridize with a complementary nucleotide sequence in another nucleic acid, RNA or DNA, to inhibit the function or synthesis of said nucleic acid. Because of their ability to hybridize with both RNA and DNA, antisense compounds can interfere with gens expression at the level of transcription, RNA processing or translation.
Antisense compounds can be designed and synthesized to prevent the transcription of specific genes to RNA by hybridizing with genomic DNA and directly or indirectly inhibiting the action of RNA polymerass. An advantage of targeting DNA is that only small amounts of antisense compounds are needed to achieve a therapeutic effect. Alternatively, antisense compounds can be designed and synthesized to hybridize with RNA to inhibit post-transcriptional modification (RNA processing) or protein synthesis (translation) mechanisms. Exemplary target RNAs are messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA) and the like. Examples of processing and translation mechanisms include splicing of pre-mRNA to remove introns, capping of the 5' terminus of mRNA, hybridization arrest and nuclease mediated mRNA hydrolysis.
At the present time, however, the development of practical scientific and therapeutic applications of antisense technologies is hampered by a number of technical problems. Klausner, A., Biotechnology, 8:303-304 (1990). Synthetic antisense molecules are susceptible to rapid degradation by nucleases that exist in target cells. The oligonucleotide sequences of antisense DNA or RNA, for example, are destroyed by exonucleases acting at either the 5' or 3' terminus of the nucleic acid. In addition, endonucleases can cleave the DNA or RNA at internal phosphodiester linkages between individual nucleotides. As a result of such cleavage, the effective half-life of administered antisense compounds is very short necessitating the use of large, frequently administered dosages.
Another problem is the extremely high cost of producing antisense compounds using available semiautomatic nucleic acid synthesizers. It has recently been estimated that the cost of producing one gram of antisense DNA is about $100,000. Armstrong, L., Business Week, Mar. 5, 1990, page 89.
A further problem relates to the delivery of antisense agents to desired targets within the body and cell. Antisense agents targeted to genomic DNA must gain access to the nucleus (i.e. the agents must permeate the plasma and nuclear membrane). The need for increased membrane permeability (increased hydrophobicity) must be balanced, however, against the need for aqueous solubility (increased hydrophilicity) in body fluid compartments such as the plasma and cell cytosol.
A still further problem relates to the stability of antisense agents whether free within the body or hybridized to target nucleic acids. Oligonucleotides such as antisense DNA or RNA are susceptible to unstable steric reconfiguration around chiral phosphate centers.
Gene targeting via antisense agents is the inevitable next step in human therapeutics. Armstrong, supra at 88. The successful application of antisense technology to the treatment of disease however, requires finding solutions to the problems set forth above. The present invention provides compounds, compositions and methods for inhibiting nuclease degradation of antisense compounds.